Published online before print January 22, 2008
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,
,1
* Departments of Anesthesiology,
Surgery,
Medicine, and
Pathology/Immunology, Division of Laboratory and Genomic Medicine, Washington University School of Medicine, Saint Louis, Missouri, USA
1 Correspondence: Department of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8054, St. Louis, MO 63110, USA. E-mail: hotch{at}wustl.edu
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80% and 85%, respectively) occurred in MyD88–/– mice. Despite the marked decrease in sepsis-induced apoptosis, MyD88–/– mice had a worsened survival. In conclusion, lymphocyte death in sepsis likely involves multiple pathogen-sensing receptors and redundant signaling pathways. MyD88 was effective in blocking apoptosis, as it is essential in mediating most pathogen recognition pathways; however, MyD88 is also critical for host survival in a model of severe peritonitis.
Key Words: cell death • cytokines • cell signaling • endotoxin
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750,000 individuals develop sepsis, and 30% succumb to this disorder annually [1
]. Sepsis is the 10th leading cause of death, and the annual financial burden incurred is in excess of $16 billion [1
]. This highly lethal disorder induces extensive apoptosis in cells of the innate and adaptive immune system, i.e., dendritic cells, B cells, and T cells [2
3
4
5
]. Morbidity and mortality in sepsis are thought to be, at least in part, a result of the loss of critical immune effector cells [6
7
8
9
]. These cells are constantly bombarded by myriad stimuli, and the integrated response to these stimuli determines the fate of the cell (e.g., activation, maturation, anergy, apoptosis). Cells of the innate and adaptive immune system have a complex repertoire of receptors, known as TLRs, which recognize and bind molecular patterns that are conserved across a variety of microorganisms [10 , 11 ]. For example, the TLR4/myeloid differentiation protein 2 complex signals upon binding LPS from Gram-negative bacteria, whereas TLR2 signals upon binding peptidoglycan from Gram-positive bacteria [12 , 13 ]. TLR2 and TLR4 transduce signals through the activation of a series of post-receptor signaling proteins, beginning with the proximal adaptor protein, MyD88. It is an essential adaptor molecule of the TLR and IL-1R family members [14 , 15 ] and has been found to be critical for the development of innate and adaptive immunity [16 ]. The association of the N-terminal death domain and C-terminal Toll/IL-1R (TIR) domain of MyD88 with the TIR domain of TLRs and IL-1R recruits downstream signaling, which leads to MyD88-dependent signaling pathways [17 , 18 ] (Fig. 1A ). This pathway involves the MAPKs, whose members include JNK, p38, and ERK1/2. MAPKs also play a role in T cell ligation, costimulatory signaling, and pro- and antiapoptotic responses. Generally, ERK1/2 activation induces antiapoptotic signals, whereas JNK and p38 mediate the proapoptotic program [19 ].
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Figure 1. Pathogen-induced cell signaling involving TIR pathways. A variety of genetic constructs was used to investigate the effects of deleting critical sites of pathogen receptor and signaling cascades. (A) MyD88-dependent signaling pathway. (A) MyD88-independent signaling pathway. The specific knockout mice (MyD88–/–, TLR2–/–, TLR4–/–, TRIF–/–, IL-1R–/–, JNK1–/–, STAT1–/–, and STAT4–/–) used are identified in red. IRAK1/4, IL-1R-associated kinases 1 and 4; TRAF6, TNFR-associated factor 6; TRIF, TIR domain-containing adaptor-inducing IFN-β; IRF-3, IFN regulatory factor 3.
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The objective of the present study was to determine the role of selective components of pathogen recognition and signaling involved in mediating the response to sepsis. Specifically, we evaluated the role of pathogen-associated molecular pattern recognition (TLR2, TLR4, and IL-1R), proximal and distal signaling molecules (MyD88, TRIF, STAT1, and STAT4), and the MAPK signaling pathway (JNK1) in sepsis-induced apoptosis by using gene-targeted deletions. Lymphocyte apoptosis was measured by two independent methods: TUNEL and intracellular signaling for active caspase 3. Given the reported role of cytokines in apoptosis and the important regulating effect of MyD88 on cytokine production in activated cells [16 ], pro- and anti-inflammatory mediator production was also analyzed in the different genetic constructs.
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Polymicrobial intra-abdominal sepsis
Mice were housed for a minimum of 1 week prior to manipulations. Polymicrobial intra-abdominal sepsis was induced by the modified CLP model of sepsis [25
]. Previously, we have reported that mice that have undergone CLP exhibit positive blood cultures for polymicrobial organisms (aerobic and anaerobic bacteria); however, sham-operated mice do not [26
]. Mice were anesthetized with halothane, and an abdominal incision was performed. The cecum was identified, ligated, and punctured with a 27-gauge needle. The abdomen was closed in two layers, and 1 ml 0.9% saline was administered s.c. Sham-operated mice were treated identically, except the cecum was not ligated or punctured. The Animal Studies Committee at Washington University School of Medicine approved animal experimentation.
Additionally, survival studies were performed with MyD88–/– and age-matched control mice. Three hours post-sham or CLP operation, mice received 25 mg/kg imipenem and twice per day thereafter for 2 additional days. Survival was recorded for 7 days.
Flow cytometry analysis of apoptosis by active caspase-3 and TUNEL
Approximately 20 h after sham or CLP operation, thymi and spleens were disassociated. To reduce nonspecific immunofluorescent staining, the cells were incubated with purified anti-mouse CD16/32 antibody (FcyRI/II) for 10 min. The thymocytes (1x106cells) were labeled with FITC-conjugated hamster anti-mouse CD3e (CD3
chain) mAb or Armenian hamster IgG isotype control (eBiosciences PharMingen, San Jose, CA, USA); splenocytes (1x106) were colabeled with FITC anti-CD3e and PE-Cy5 CD45R/B220 or Armenian hamster IgG isotype control or PE-Cy5 rat IgG2a isotype control (eBiosciences PharMingen) for 30 min as described previously [27
]. Following labeling, cells were washed with PBS and fixed in 2% paraformaldehyde for 30 min at room temperature. Next, cells were permeabilized with 90% methanol for 30 min on ice in the dark and washed with PBS. Afterwards, cells were stained with TUNEL (Phoenix Flow Systems, Inc. San Diego, CA, USA) for 2 h at 37°C or active caspase 3 (Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C, as per the manufacturer’s directions and as described previously [27
]. The cells were washed with PBS and analyzed (25,000–50,000 events/sample) on FACScan (BD Biosciences, San Jose, CA, USA) using gates set with appropriate controls. CELLQuestTM software was used to analyze apoptosis.
Cytokine analysis
Simultaneous measurement of serum cytokine levels of IL-6, IL-10, IFN-
, TNF, and IL-12p70 was performed on the BDTM Cytometric Bead Array Mouse Inflammation kit (BD Biosciences), as described previously [28
]. Reagent dilutions were prepared according to the manufacturer’s specifications. Standards, samples, capture beads, and PE detection reagent (50 µl) were added to the appropriate wells in a filter-bottom, 96-well microtiter plate, which was shaken for 5 min at 500 rpm and incubated for 2 h at room temperature in the dark. Lastly, wash buffer was added to all the wells, the microtiter plate was shaken for 5 min at 500 rpm to resuspend the capture beads, and cytokine levels were quantified on the FACSArray (BD Biosciences). Limits of detection for the cytokines are as follows: IL-6 (5.0 pg/ml), IL-10 (17.5 pg/ml), IFN- (2.5 pg/ml), TNF (7.3 pg/ml), and IL-12p70 (10.7 pg/ml).
Statistical analysis
Data were analyzed with the statistical software Prism (GraphPad). Data are reported as the mean ± SEM. The Student’s t-test was used in analyzing data involving only two groups. One-way ANOVA with Tukey’s multiple comparison tests was used to analyze data with more than two groups. Kruskal-Wallis and the Mann-Whitney post-test were used for nonparametric analysis. Significance was reported at P 
0.05.
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Figure 2. Analysis of apoptosis via flow cytometry of thymocytes from MyD88–/– and wild-type mice subjected to CLP or sham surgery. (A) The unstained control and the isotype control exhibit fluorescence profiles that are indistinguishable from each other but different from the positive control anti-CD3. The quadrants were set based on the negative controls. (B) Apoptosis was determined by TUNEL staining. The percentage of cells in each quadrant is identified in parentheses. The percentage of CD3+ T cells undergoing apoptosis is identified in the upper right-hand quadrants. Limited apoptosis was exhibited in MyD88–/– mice with sepsis (CLP) and sham-operated mice. However, wild-type mice exhibited a significant increase in apoptosis compared with all groups; n = 1 mouse for each flow diagram.
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Figure 3. Thymocytes and splenocytes from MyD88–/– mice have decreased sepsis-induced apoptosis. T and B lymphocytes from MyD88–/– mice are protected from sepsis-induced apoptosis as determined by active caspase-3 and TUNEL staining. MyD88–/– mice underwent CLP or sham surgery and were killed 20 h following surgery. Apoptosis was quantitated by active caspase-3 and TUNEL. Lymphocyte apoptosis in MyD88–/– mice with sepsis (CLP) was not different from sham-operated mice. However, apoptosis (active caspase-3 and TUNEL) in MyD88–/– mice with sepsis (CLP) was significantly less compared with wild-type mice (WT). *, P < 0.05, MyD88–/– mice versus wild-type CLP (n=9 for MyD88–/– mice, and n=9 for wild-type mice).
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Figure 4. H&E staining of thymi and spleens. Qualitative light microscopic evidence of MyD88–/– mice splenocyte protection against sepsis-induced apoptosis (600x magnification). Thymocytes from wild-type mice with sepsis show extensive apoptosis, and virtually all cells demonstrate hallmarks of apoptosis, including compacted and fragmented nuclei. In contrast, the majority of thymocytes from the MyD88–/– mice appears normal with reduced compaction and fragmentation. Similarly, focal areas of splenocytes from the wild-type mice with sepsis demonstrate nuclear injury (identified by circles), and only rare splenocytes from MyD88–/– mice show apoptotic changes.
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Figure 5. Thymocytes and splenocytes from TLR2–/– mice exhibit sepsis-induced apoptosis. T and B lymphocytes from TLR2–/– mice are not protected from sepsis-induced apoptosis as determined by active caspase-3 and TUNEL staining. TLR2–/– mice underwent CLP or sham surgery and were killed 20 h following surgery. Apoptosis was quantitated by active caspase-3 and TUNEL. Lymphocyte apoptosis in TLR2–/– mice with sepsis (CLP) was significantly different from sham-operated mice (P<0.05). However, lymphocyte apoptosis in TLR2–/– mice with sepsis (CLP) was not different from wild-type mice with sepsis (CLP; n=9 for TLR2–/– mice, and n=9 for wild-type mice).
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Figure 6. Thymocytes and splenocytes from TLR4–/– mice exhibit sepsis-induced apoptosis. T and B lymphocytes from TLR4–/– mice are not protected from sepsis-induced apoptosis as determined by caspase-3 and TUNEL staining. TLR4–/– mice underwent CLP or sham surgery and were killed 20 h following surgery. Apoptosis was quantitated by active caspase-3 and TUNEL. Lymphocyte apoptosis in TLR4–/– mice with sepsis (CLP) was significantly different from sham-operated mice (P<0.05). However, lymphocyte apoptosis in TLR4–/– mice with sepsis (CLP) was not different from wild-type mice with sepsis (CLP; n=9 for TLR4–/– mice, and n=9 for wild-type mice).
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Figure 7. Thymocytes and splenocytes from TRIF–/– mice exhibit sepsis-induced apoptosis. T and B lymphocytes from TRIF–/– mice are not protected from sepsis-induced apoptosis as determined by active caspase-3 and TUNEL staining. TRIF–/– mice underwent CLP or sham surgery and were killed 20 h following surgery. Apoptosis was quantitated by active caspase-3 and TUNEL. Lymphocyte apoptosis in TRIF–/– mice with sepsis (CLP) was significantly different from sham-operated mice (P< 0.05). However, lymphocyte apoptosis in TRIF–/– mice with sepsis (CLP) was not different from wild-type mice with sepsis (CLP; n=9 for TRIF–/– mice, and n=9 for wild-type mice).
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in wild-type mice with sepsis (Table 1
). Compared with wild-type mice with sepsis, MyD88–/– mice showed a significantly impaired Th1 cytokine response; i.e, the abundance of circulating IL-6, TNF, and IL-12p70 was decreased by 95% (Table 1)
. IFN- and IL-10 were elevated in septic wild-type mice, but they were below the limit of detection in MyD88–/– mice (Table 1)
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Table 1. Circulating Cytokines in Various Genetic Constructs with Sepsis
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or IFN- (reviewed in ref. [23
]), exhibited a significant attenuation of IL-6, IFN-, and IL-10 levels compared with wild-type mice (Table 1)
. As expected, STAT4–/– mice, which are unable to respond to IL-12 (reviewed in ref. [22
]), had significantly decreased IL-12 and IFN- production (Table 1)
. Although, TNF and IL-6 levels trended to be higher in the wild-type mice compared with TLR4–/– and STAT4–/– mice and TLR2–/– and TLR4–/– mice, respectively, these results did not reach statistical significance (Table 1)
.
MyD88–/– mice have increased mortality in sepsis
To determine the overall beneficial versus the adverse effect of decreased sepsis-induced lymphocyte apoptosis and the altered pro- and anti-inflammatory cytokine production in MyD88–/– mice, survival studies were conducted. Compared with septic wild-type mice, septic MyD88–/– mice had a marked decrease in survival, 62.5% versus 12.5%, respectively (P<0.05; Fig. 8
; n=8 for each group).
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Figure 8. MyD88–/– mice have increased mortality in sepsis. MyD88–/– mice and age- and sex-matched wild-type mice underwent CLP to induce sepsis. Survival was followed for 7 days. MyD88–/– mice had an increased mortality compared with wild-type mice; *, P < 0.05; n = 8 for wild-type mice, and n = 8 for MyD88–/– mice.
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Our findings are consistent with the work of Dear et al. [35 ], who examined the role of MyD88 in a model of acute renal failure induced by polymicrobial sepsis (CLP model). Although these authors did not examine individual cell phenotypes, they did report that spleens from MyD88–/– mice with sepsis exhibited significantly reduced apoptosis compared with spleens of wild-type mice with sepsis. However, these authors did not report the effects of sepsis on survival in MyD88–/– mice. Our present study is an extension of the work of Dear and associates [35 ] and characterizes the protection elicited during polymicrobial sepsis in defined lymphocyte populations of the thymus (T lymphocytes) and spleen (T and B lymphocytes) via flow cytometry. In addition, the impact of MyD88 deletion on sepsis survival was recorded [35 ]. MyD88 is a central mediator of the cellular response involved in pathogen recognition. Virtually all members of the TLR family use MyD88 for signaling. A key function of the TLRs is to induce a cytokine response to activate the host immune defenses. MyD88–/– mice showed abrogated mediator production compared with wild-type mice, and this decrease in cytokines may be one mechanism responsible for the decrease in apoptosis in T and B lymphocytes.
A key question is whether lymphocytes from MyD88–/– mice were protected from sepsis-induced apoptosis because of intrinsic or extrinsic factors. B and T cells are known to possess specific TLR receptors including TLR2 and TLR4 [36
37
38
]. Therefore, under normal conditions, B cells [39
] and some T cells express MyD88 [40
], which is a key component of TLR signaling. Isolated, splenic T cells from wild-type mice underwent RT-PCR for mRNA for TLR2 and TLR4 and were positive for both receptors (data not shown). Thus, deletion of MyD88 could be an "intrinsic" mechanism of protection. Another potential mechanism for attenuation of lymphocyte apoptosis in MyD88–/– mice may be alterations in circulating cytokines or chemokines. A number of cytokines, e.g., TNF- and IFN-, are reported to induce lymphocyte cell death in specific settings. The concentration of circulating cytokines was dramatically reduced in MyD88–/– mice. Therefore, a second potential mechanism for the decrease in sepsis-induced lymphocyte apoptosis in MyD88–/– mice is a result of extrinsic environmental factors, e.g., the modulation of proapoptotic cytokines or chemokines. Although we are not able to definitively distinguish which of these two potential mechanisms is responsible for the protection against apoptosis in sepsis, preliminary studies from our laboratory showed that both mechanisms may be involved in the protection against sepsis-induced apoptosis. The studies were technically difficult because of limitations in recovering a sufficient number of labeled cells that had been adoptively transferred. The studies suggested, however, that lymphocytes from wild-type mice that were adoptively transferred into MyD88–/– mice with sepsis had a marked reduction in cell death, and lymphocytes from MyD88–/– mice that were adoptively transferred into wild-type mice with sepsis also had decreased apoptosis (unpublished observations). These finding are consistent with protection in lymphocytes in MyD88–/– being a result of a decrease in extrinsic factors of the host milieu, such as decreased circulating cytokines/chemokines as well as defects in the apoptotic signaling cascade.
It is interesting that deletion of MyD88 prevented sepsis-induced lymphocyte apoptosis, but deletion of TLR2 or TLR4, proximal pathogen-sensing receptors, failed to do so. Deletion of TLR4 failed to block sepsis-induced apoptosis and is consistent with earlier studies from our laboratory showing that the mice with a defective TLR4 receptor, i.e., C3H/HeJ mice, had no decrease in sepsis-induced lymphocyte apoptosis [41 ]. The CLP model of sepsis is a polymicrobial infection consisting of Gram-positive bacteria and Gram-negative bacteria and likely leading to engagement of multiple TLRs. Furthermore, recent studies found that a single individual bacterium can activate multiple TLRs [12 ]. Similar to deficiency of TLRs, knockout of IL-1R also failed to result in diminished sepsis-induced lymphocyte apoptotic death. Considered together, the data suggest that deletion of a single proximal receptor such as TLR2, TLR4, or IL-1R will not reduce apoptosis in sepsis, as redundant signaling pathways are activated in this complex disease.
The signal mediated by TLR4 activation not only occurs via MyD88 but also by a second MyD88-independent pathway mediated by TRIF (Fig. 1) . Overexpression of TRIF increases cell death in response to specific noxious stimuli [42 ], and TRIF–/– mice have been reported to exhibit reduced, LPS-induced dendritic cell apoptosis. Therefore, we examined the role of TRIF in sepsis-induced lymphocyte apoptosis. Deletion of TRIF did not ameliorate sepsis-induced T or B cell death, and furthermore, mediator production in TRIF–/– mice did not differ significantly from wild-type mice (Table 1) .
Inconsistencies in the literature concerning the MAPK JNK have spawned controversy as to whether it alone has proapoptotic or antiapoptotic effects [43 ]. JNK-induced apoptosis is cell type- and stimulus-specific. JNK1–/– mice are developmentally normal and show normal T and B cells [44 ]. In our CLP model of sepsis, lymphocyte apoptosis was not ameliorated in JNK1–/– mice. Although JNK1–/– mice were deficient in JNK1, they had fully functional JNK2 genes [45 ]. The lack of lymphocyte protection may be explained by redundant activation of other MAPKs, such as JNK2 or p38, which participate in proapoptotic responses. Furthermore, JNK2 has been implicated in preferentially up-regulating Fas ligand and caspase 3 expression [46 , 47 ]. We did not examine the effects of multiple JNK gene deletions, as deletion of JNK1 and JNK2 genes results in embryonic lethality [44 ]. Similar to the findings in the JNK1–/– mice, there was no diminution in sepsis-induced apoptosis in Stat1–/– and Stat4–/– mice, and this lack of protection may again be a result of overlapping signaling pathways.
Despite the dramatic reduction in sepsis-induced lymphocyte apoptosis in MyD88–/– mice, there was a seemingly paradoxical increase in sepsis mortality. This increase in septic-induced mortality in the MyD88–/– mice is not surprising, given the profound decrease in the ability of these mice to mount an effective immune response. As the MyD88 pathway is essential for allowing the host to sense invading pathogens, these mice produce virtually no pro- or anti-inflammatory cytokines that are required to combat the microorganisms. It is this unresponsiveness to bacterial products that renders MyD88–/– mice resistant to endotoxin lethality [48 ] but more susceptible to death from live, infectious organisms such as Streptococcus pneumoniae [49 ]. Local and systemic inflammatory responses to S. pneumoniae depend on the presence of MyD88 to clear bacterial colonization of the upper respiratory tract and to prevent pulmonary and systemic infection in mice [49 ]. Numerous researchers have reported that MyD88–/– mice are extremely susceptible to monomicrobial infections. However, to our knowledge, our in vivo findings that demonstrate MyD88–/– mice exhibit worse survival during polymicrobial sepsis (CLP model) are novel. Interestingly, Weighardt et al. [50 ] reported that MyD88–/– mice have improved survival in acute polymicrobial peritonitis in their model of colon ascendens stent peritonitis (CASP). The improved survival noted in the MyD88–/– mice in the CASP model may be a result of the fact that CLP and CASP have distinct pathophysiologic effects [51 ] with variations in pathogen kinetics. MyD88–/– mice would tend to be protected in acute fulminant models of sepsis, in which deaths are a result of a "cytokine storm". In contrast, MyD88–/– mice would tend to be more susceptible to sepsis of slower onset, in which the deaths are a result of failure to control/eradicate the invading pathogens. Thus, a difference in the pathologic mechanisms may explain the discrepancy in the two models in MyD88–/– mice. Furthermore, the diminished mediator production and worsened survival exhibited in our MyD88–/– mice may, in part, be a result of incomplete expansion of GR-1+ CD11b+ cells. Delano et al. [52 ] reported GR-1+ CD11b+ cells play a MyD88-dependent role in sepsis-induced T lymphocyte suppression and Th2 polarization.
Only the deletion of MyD88 and not other receptors or signaling molecules prevented sepsis-induced lymphocyte apoptosis. The explanation for this finding is most likely because lymphocyte death in sepsis involves multiple pathogen-sensing receptors and redundant signaling pathways; therefore, it is necessary to inhibit proximal adaptor proteins, e.g., MyD88, which transduce signals from multiple receptors. The present work about MyD88 is analogous to previous studies from our laboratory, in which we demonstrated that lymphocytes from mice with a dominant-negative Fas-associated death domain were protected from sepsis-induced apoptosis, but lymphocytes from mice with deletion of death receptors (Fas and TNF) were not protected [28 , 53 ]. In conclusion, lymphocyte death in sepsis is mediated by multiple pathogen-sensing receptors and redundant signaling pathways. Blocking lymphocyte death in sepsis requires inhibition at a site that is common and proximal to multiple pathogen-sensing/signaling pathways. Caution is necessary in inhibiting these pathways, as such inhibition may be detrimental to the ability of the host to respond to pathogenic microorganisms.
Received August 9, 2007; revised December 7, 2007; accepted December 12, 2007.
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B and IFN-regulatory factor 3, in the Toll-like receptor signaling J. Immunol. 171,4304-4310 is required for activation-induced death of T lymphocytes J. Exp. Med. 196,999-1005, but not MyD88, regulate Escherichia coli-induced dendritic cell maturation and apoptosis in vivo J. Immunol. 175,839-846This article has been cited by other articles:
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  R. J. Freishtat, J. Natale, A. S. Benton, J. Cohen, M. Sharron, A. A. Wiles, W.-M. Ngor, B. Mojgani, M. Bradbury, A. Degnan, et al. Sepsis Alters the Megakaryocyte-Platelet Transcriptional Axis Resulting in Granzyme B-mediated Lymphotoxicity Am. J. Respir. Crit. Care Med., March 15, 2009; 179(6): 467 - 473. [Abstract] [Full Text] [PDF]
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